My research program at URI can be described by four related projects:

1. Inhibitors of human sulfotransferases
2. Biotransformation of indole to the uremic toxin indoxyl sulfate
3. In vivo metabolism of the food-borne carcinogen 2-amino-alpha-carboline
4. Effect of sulfotransferase inhibitors on estradiol homeostasis and actions

Project 1: Reversible inhibitors of human sulfotransferases
Sulfotransferase enzymes catalyze the conjugation of endogenous compounds including monoamine neurotransmitters, steroid hormones and thyroid hormones. They also sulfonate a wide variety of xenobiotic substrates including many therapeutic drugs, dietary constituents, and environmental compounds. Depending on the substrate, the result of sulfonate conjugation leads to one of three results: (1) decrease in biological/therapeutic activity, (2) increase in biological/therapeutic activity, or (3) increase in cytotoxicity or genotoxicity via covalent adduct formation from a reactive metabolite. Thus, inhibition of sulfotransferase activity can lead to either beneficial or adverse effects depending on the substrate and isoform involved. Since sulfotransferases are involved in so many different biological processes, it is imperative to understand mechanisms modulating the activity of each sulfotransferase isoform.

The specific aims of this project are to (1) identify inhibitors of the important human cytosolic sulfotransferases by screening selected environmental and dietary compounds, and (2) use a structure-guided design approach to develop inhibitors which are highly specific for each sulfotransferase member. We select compounds for screening based on structural relationships with known sulfotransferase ligands. Since inhibitors identified by screening are of varying specificity and may affect other enzyme systems or steroid receptors, there is also a critical need for a structure-based design approach to elucidate the structural features necessary for potent and specific binding to the substrate/cofactor binding site for each major sulfotransferase isoform. A portion of aim 1 of this project is complete and recently submitted for publication. Aim 2 is the subject of current research and proposals pending.

• Presentations: 1 invited oral (2002), 2 posters with published abstracts (2000, 2001), 1 additional poster (2001), and many student presentations

• Publications: 1 total, Current Drug Metabolism, submitted September 21, 2005

• 1 M.S. thesis.

• Funding: 5 awards, total of $155,980; 1 pending

• Personnel: Anasuya Ghosh, Jinfang Wu, Nicholas Rue

• Status of project: continuing

Project 2: Biotransformation of indole to indoxyl sulfate
Indoxyl sulfate, a sulfate conjugate of indoxyl, is a known circulating uremic toxin promoting the progression of glomerular necrosis and renal failure, and serum concentration of this metabolite is markedly increased in uremic patients. Indoxyl sulfate is mainly produced after bacterial decomposition of dietary tryptophan in the intestines. The initial product of tryptophan metabolism in the intestines is indole, which is subsequently hydroxylated to indoxyl and sulfonated to form indoxyl sulfate. It had been speculated that the initial tryptophan metabolite indole is absorbed, hydroxylated by cytochrome P-450 (P450, CYP) enzymes, conjugated by sulfotransferases (SULT), and excreted by the kidneys. The goal of this study was to determine which isoforms of cytochrome P450 and sulfotransferase were responsible for human metabolism of indole to indoxyl sulfate.

In brief, we found that human and rat cytochrome P450 enzymes catalyze oxidation of indole to indoxyl and that CYP2E1 isoform is responsible for indoxyl formation in rat liver microsomes. Since the human CYP2E1 is very similar catalytically to the rat CYP2E1, we proposed that human CYP2E1 is the isoform responsible for indoxyl formation in humans. We also found that human and rat phenol sulfotransferases catalyze sulfonation of indoxyl to indoxyl sulfate and that SULT1A1 isoform is responsible for the sulfonation activity in human liver cytosol. Since both CYP2E1 and SULT1A1 exhibit wide individual variation in activity, these results support a mechanism for individual variation in formation of a toxic metabolite and may causes differences in susceptibility to the toxic effects of indole and indoxyl sulfate formed from dietary tryptophan.

• Publications: 2 total, European Journal of Drug Metabolism and Pharmacokinetics, 2000 and 2001.

• Funding: 1 award, $3,000 total

• Personnel: Erden Banoglu

• Status of project: complete

Project 3: Metabolism of the heterocyclic amine mutagen 2-amino-alpha-carboline
2-Amino-alpha-carboline (AaC) is one of the five major heterocyclic aromatic amines (HAAs) present in the diet and is formed by pyrolysis of tryptophan. HAAs are naturally occurring genotoxic carcinogens produced by cooking meat and other protein-containing foods. They are not present in uncooked meat, but are readily produced under normal household cooking conditions. In addition to dietary exposure, AaC is found in combustion smokes of wood and cigarettes, in automobile exhaust, and in municipal water sources. Human exposure to HAAs including AaC is low but chronic. AaC is clearly mutagenic and carcinogenic in model systems. For example, AaC induced preneoplastic lesions in the livers of male F344 rats, mutations in the colon of transgenic Big Blue mice, and lymphomas and liver tumors in CDF1 mice. However, despite many years of study, association of heterocyclic amine exposure with human carcinogenesis remains suspected but unproven.

The aim of this study is to use model systems (tissue homogenates, purified enzymes, cultured cells, rodents in vivo) to develop methods for efficient detection, identification and quantification of metabolites and DNA adducts of the heterocyclic amine 2-amino-alpha-carboline. Our goal is to develop plausible biomarkers for human molecular epidemiological and exposure studies for AaC. These are necessary to clarify whether AaC is a human carcinogen at the low but chronic human exposures.

Our results show that AaC is highly metabolized by oxidation and conjugation to stable, excreted metabolites. Approximately 14 metabolites were observed in the rat bile from the in vivo study, and initial structure determination indicated oxidation and extensive conjugation. Further structure elucidation was conducted on a similar number of metabolites formed and excreted by rat hepatocytes and human hepG2 liver tumor cells. We found three sites of aromatic ring hydroxylation, one more than observed in the microsomal studies we published previously. Four major metabolites were formed in both cell systems, three of them identical: AaC-6-sulfate, AaC-3-sulfate, and N-acetyl-AaC. In human liver tumor cells (hepG2) the fourth major metabolite was a double conjugate, N-acetyl-AaC-6-sulfate. In rat hepatocytes, the fourth major metabolite was one of the AaC-N-glucuronides. We confirmed expected sites of AaC metabolism, but also observed unexpected metabolites. The unexpected metabolites include extensive N‑acetylated conjugates and a total of three different N-glucuronides. Also noteworthy are metabolites that were not detected: no direct N-sulfonation to form the sulfamate, and very little O‑glucuronidation even by the rat hepatocytes which are known to have active glucuronidation systems.

These results combined with our earlier publications on the reactive, DNA-adduct-forming, metabolites of AaC indicate that both bioactivation and detoxification share the same metabolic pathways—oxidation, acetylation, and sulfonation. We have not yet confirmed the isoforms responsible for transformation to the stable metabolites, but based on other HAAs it is likely to be CYP1A2, SULT1A1, NAT1, and NAT2. These are the same enzymes responsible for the DNA-reactive metabolites. This may have the end result of ‘protecting’ those individuals who have relatively high levels of these enzymes—increased bioactivation would be balanced by increased detoxification.

• Presentations: 2 invited oral (2001, 2004), 2 posters with published abstracts (2002, 2004), 2 additional posters (2002, 2004), and many student presentations

• Publications: 3 total (1999, 2000, 2000), 2 in preparation

• 1 M.S. thesis.

• Funding: 4 awards, total of $140,980

• Personnel: Gautam Jha, Zhixin Yuan

• Status of project: complete

Project 4: Effect of sulfotransferase inhibitors on estradiol homeostasis and actions
Increased estrogen exposure is clearly related to mammary cancer development, but mechanisms controlling mammary tissue estradiol homeostasis are only partially understood. Several enzymes are known or proposed to be involved in the control of local estradiol biosynthesis and degradation including aromatase, sulfatase, 17b-hydroxysteroid dehydrogenase, and estrogen sulfotransferase (EST). However, little is known about the role of sulfotransferase toward estradiol homeostasis in normal or transformed human mammary cells. Because of the abundance of environmental and dietary compounds known to be relatively potent inhibitors of EST, we believe it is imperative to understand the (potentially negative) cellular effect of these inhibitors on estradiol concentrations and downstream actions.

Our long-term goals are to establish the relative contribution of EST toward regulation of breast cellular estrogen activities in cancer initiation and progression, and to establish the mechanisms of EST suppression and stimulation in breast cancer. The central hypothesis of this study is that EST inhibition/suppression will directly alter endogenous cellular estradiol concentrations and mimic higher estradiol exposure in the cell model systems. As models, we are studying the effect of EST inhibition on transformed (MCF-7) and non-transformed (human mammary epithelial, HME) cell systems. These two systems will establish the approach toward both initiation (HME) and progression (MCF-7). Our approach is to modulate EST activity in the cell systems and, directly and indirectly, measure the effect on local estradiol concentration and estrogenic activities.

This project is innovative in that it is the first to directly relate change in local estradiol concentration with activity of estradiol metabolic enzymes in human mammary cell systems. Moreover, it focuses on pharmacological inhibitors of human estrogen sulfotransferase. Many environmental and dietary compounds are known to be relatively potent inhibitors of EST, but no one has shown whether these compounds actually alter cellular estradiol concentrations or affect estrogenic activity. The rationale for this work is that fuller understanding of the effect of EST inhibition on normal and transformed human mammary cell systems may establish EST inhibition as an additional mechanism for mammary cancer initiation or progression.

• Presentations: regional and student presentations

• Publications: 1 in preparation

• 1 M.S. thesis.

• Funding: 3 awards, total of $34,150

• Personnel: Jinfang Wu, Roseanne Meyer

• Status of project: continuing 

Collaborative Project 5: Metabolism of benzo[ghi]fluoranthrene: determination of microsomal metabolites (with Bongsup P. Cho)
Two undergraduate students have worked on this project during one summer each. This compound is a carcinogenic PAH under study by Bongsup Cho. The goal of this work was to profile the rat liver microsomal metabolites of benzo[ghi]fluoranthrene. The first student found the metabolites formed by microsomal oxidation systems were not the same as the synthetic oxidation products. (Their HPLC retention time and UV/Vis spectra were different.) This result is very interesting and of potential scientific impact. A second student scaled-up the microsomal incubate enough to allow preparation of enough compound for structure determination. His samples are awaiting further purification and structure analysis.

• Presentations: student presentations

• Publications: none

• Funding: none

• Personnel: Siobhan O’Brian (1 summer), Steve Rougas (1 summer)

• Status of project: inactive 

Collaborative Project 6: Metabolism of linolenic acid in salmon liver hepatocytes (with Chong Lee, Nutrition)

The goal of this project is to determine how various treatments affect relative formation of known linolenic acid metabolites (DHA, E) in salmon. I am supervising Mary Anne Eaton (graduate student in Chong Lee’s lab) in hepatocyte isolation, incubation with radiolabeled substrate, and subsequent analysis and quantitation of metabolites. This project is similar in technique and approach to my own work on metabolism of heterocyclic aromatic amine carcinogens.

• Presentations: none

• Publications: none

• Funding: by Chong Lee only

• Personnel: Mary Anne Eaton (graduate student in Nutrition)

• Status of project: continuing

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